PROJECT SUMMARY Emerging evidence has uncovered a critical role for cellular metabolism as stem cells (SCs) balance self- renewal and differentiation to maintain tissue homeostasis. However, little is known mechanistically about how SCs integrate metabolic stimuli during cell fate decisions within their in vivo niches. Furthermore, several classes of metabolites are increasingly being recognized as able to influence epigenetic processes that control chromatin remodeling, including highly plastic chromatin domains that direct key transcriptional programs to maintain SC identity. Thus, achieving a better understanding of the complex crosstalk between metabolism, epigenetics and SC function is critical to identify essential signaling cascades necessary to preserve SC- mediated regenerative capacity. To achieve these goals, the murine hair follicle is an excellent system because it naturally undergoes recurrent and synchronous cycles of tissue homeostasis and regeneration. First, I will isolate and purify populations of hair follicle stem cells (HFSCs) during quiescence, activation and following their transition to short-lived, proliferative progenitors. I will then perform in vivo metabolomic profiling by directly measuring metabolite abundance using gas chromatography?mass spectrometry. Stable isotope tracing will determine metabolite turnover rates and their relative contributions from several carbon sources, including glucose sugars, fatty acids and the amino acid glutamine (Aim I). Next, I will engineer bioenergetic sensors to monitor the metabolic dynamics of HFSCs throughout the hair cycle and within in vivo physiological setting. This will be accomplished by exploiting my lab's powerful and rapid in utero lentiviral technology to deliver these sensors specifically to the skin epithelium and hair follicle (Aim II). I will apply CRISPR-mediated knockout strategies using the in utero lentiviral delivery platform to functionally assess the impact of metabolic perturbations to the metabolism and behavior of HFSCs. And finally, I will measure the impact of these interventions aimed at critical metabolic pathways on chromatin remodeling in HFSCs, clarifying the mechanistic contributions played by metabolic adaptations during the epigenetic switch governing the transition from self-renewal to lineage commitment and differentiation (Aim III). In total, this work will define the functional consequences and epigenetic mechanisms underlying metabolic reprogramming in the regenerating hair follicle, providing novel therapeutic targets that have the potential to help maintain long-term tissue homeostasis in diseases affected by SC imbalance, ranging from aging to cancer.